There is an urgent need to control the global epidemic of HIV infection and the development of a vaccine against HIV is one of the major objectives in AIDS research. In general vaccines should activate antigen presenting cells, overcome genetic restriction in T-cell responses and generate T- and B-memory cells. The variability of the viral population poses a further difficulty in obtaining an effective HIV vaccine. A break through in the ongoing attempts to develop a vaccine against AIDS has so far not been reported. It is now generally accepted that an induction of antigen-specific humoral and cell-mediated immunity is crucial for a development of an effective prophylactic and therapeutic vaccine. All three arms of the immune system including neutralizing antibodies; CD8+CTL and T-helper-1 (TH1) cells might be required for protective immunity to HIV. It is known that CTL can clear other viral infections (Ada, Immunol. Cell Biol., 72:447-454, 1994) and that CTL can lyse infected targets early in infection before viral progeny can be produced and released by cell lysis, Ada et al., supra. The focus has been on selection of antigens as well as on design and evaluation of different adjuvances. The antigens used in different in vitro and in vivo studies have been all from crude proteins to various synthetic peptides from several of the HIV proteins. A large number of studies have been done on the V3 loop of gp120. Induction of both B- and T-cell responses have been observed, however, it has been reported from an in vitro study that a peptide from the conserved region of gp41 have indicated infection enhancement (Bell S. J., et al., Clin. Exp. Immunol., 87 (1): 37-45, (January 1992).
Naturally occurring HIV sequences in vaccine candidates are not capable of stimulating a stable immune response due to the viruses inherent ability to hide by changing the appearance of the epitopes presented on the cell surface of infected cells. The immune system is fooled to believe that a particular amino acid sequence is relevant when in fact the amino acids of importance is hidden.
A recent study of titers of antibodies against the gag p24 protein, has shown that slow progression towards development of AIDS is associated with high titers, while fast progression towards development of AIDS is associated with low titers. It is shown that persons with low p24 antibody titer develop significantly faster AIDS than persons with high p24 antibody titers (Zwart G., et al. Virology, 201, p. 285-93, June 1994), indicating that gag and p24 in particular can play a key role to control the development of AIDS.
New HIV p24 peptides are described in WO91/13360, wherein the peptides are used in a method of discriminating between a false and true diagnosed HIV-positive serum sample.
Johnson R. P., et al., The Journal of Immunology, Vol. 147, p. 1512-1521, No. 5, Sep. 1, 1991 describe an analysis of the fine specificity of gag-specific CTL-responses in three HIV-1 seropositive individuals, the gag-specific CTL-responses were found to be mediated by CD3+ CD8+ lymphocytes which are HLA class I restricted. Goulder P. J. R. et. al., Journal of Virology, Vol. 74, p. 5679-5690, No 12, June 2000 has studied CTL response from different parts of p17 and p24 of HIV in different populations. The findings show that certain immunodominant regions exist, however, minor differences in amino acid composition can cause large differences in response.
EP-A-0 356 007 discloses antigenic determinants, in particular it relates to synthetic polypeptide sequences which are related to proteins present in the HIV-1 and which can be used as a basis for a potential vaccine against AIDS.
Rosenberg E. S. et al., Science, Vol. 278, 21 Nov. 1997, p. 1447-1450 describe that virus specific CD4+ T helper lymphocytes are critical to the maintenance of effective immunity in a number of chronic viral infections, but are characteristically undetectable in chronic human immunodeficiency virus-type 1 (HIV-1) infection. HIV-1-specific proliferative responses to p24 were inversely related to viral load. They conclude that the HIV-1-specific helper cells are likely to be important in immunotherapeutic interventions and vaccine development.
EP 0 230 222, EP 0 270 114, DE 37 11 016 and GB 2 188 639 all in the name of F. Hoffmann-La Roche & Co. Aktiengesellschaft concern recombinant expression and purification of an HTLVIII Gag/Env gene protein or fusion proteins. The proteins consisting of native sequences can be purified to homogeneity and used as a basis for diagnostic tests for detection of antibodies against viruses associated with AIDS. The gag/env protein may also be formulated for use as a vaccine for protection against AIDS through prophylactic immunization.
From a diagnostic and therapeutic point of view, the major problems with using p24 as part of an assay or therapy is associated with the high number of epitopes on p24 which stimulates production of a large number of antibodies with poor specificity, which through repeated boostering on potential mutated sequences can create autoantibodies (Autoantibodies to the alfa/beta T-cell receptors in HIV infection; dysregulation and mimicry. Lake D. F., et al., Proc. Natl. Acad. Sci. USA, (23): 10849-53, Nov. 8, 1994). Further, it is reported that the p24 antibody titer does not reach the same high levels as for the envelope proteins (gp120 and gp41). Normally antibodies to p24 are developed in the very early phase of the infection, but the titer is fairly quickly stabilized after the initial infection period. Later the p24 titer is gradually decreasing while the opposite happens with gp160. These findings can also be seen in relation to recent reports stating that cytotoxic T-cell activity is antagonized by naturally occurring HIV-1 gag variants (Klenerman P., et al., Nature, 2:369 (6479), p. 355, 2 Jun. 1994). This can be one of the reasons why a rapid stabilization of the p24 titer is seen and why it later starts to decrease.
Based on the above background data, we decided to investigate the possibility of designing novel synthetic peptides which can mimic the p17 and p24 epitopes without antagonizing the cytotoxic T-cell activity, in order to meet the need for an effective prophylactic and therapeutic vaccine.
The sequence of p17 identified as a possible template for development of peptides that can elicit CTL and antibody response is published by Korber B., et al., Human Retroviruses and AIDS 1999 Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. The identified amino acid sequence is located between the amino acids 33 and 53, confer table 1:
TABLE 1AAAA no.sequenceNaturally occurring AA's33H34ILVM35IV36W37A38SNR39RS40E41LM42EDKGQ43RKGN44FSY45ATS46VLIC47NDS48PRST49GSADNQ50LF51LM52EGD53TSA
The one letter as well as the three letter codes defining the amino acids in the sequences give throughout this specification are in accordance with International standards and given in textbooks, for instance Lehninger A. L., <<(Principles of Biochemistry>>, Worth Publishers Inc., New York, 1982. The amino acids given to the right of the second column represent the natural variation of the sequence. A change in the overall charge of the epitope by modification of amino acids can involve a significant improvement of the immunogenicity. The modifications involve a probable conformation change from the original helical to a sheet structure, exposing the epitope to the immune system in a different manner and expectingly to a greater extent.
To further increase the number of T-cell epitopes and reduce the probability for development of escape mutants within the gag protein three additional peptide sequences from p24 were based on the following three sequences from residues 133-158, 178-199 and 233-251, respectively published in Human Retroviruses and AIDS 1999; A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, confer tables 2-4:
TABLE 2AANaturally occurring AA'sAA no.sequenceat each AA position133P134IVL135VMAI136QSTV137NDT138IALM139QEKG140G141QI142MPA143VIATR144H145QH146ASVNP147ILMV148ST149PA150R151T152LS153NF154A155W156V157K158VAC
TABLE 3AANaturally occurring AA'sAA no.sequenceat each AA position178G179A180TAIVL181PS182QHGTSY183D184LIV185NY186TMLA187M188L189NST190TIVA191VI192G193GD194H195Q196AG197A198ML199QEHand
TABLE 4Naturally occurring AA'sAA no.AA sequenceat each AA position233G234SA235D236I237A238G239TAS240TS241ST242TNS243LPVQ244QAH245E246QH247ILVM248GAQTNRHI249W250MT251TS
Several modified peptides have been synthesized in order to determine unique sequences which are both specific and sensitive towards HIV-1.